PROJECT SUMMARY The development of broadly cross-reactive neutralizing antibodies (bNAbs) in HIV-1 infected individuals requires iterative rounds of envelope (Env)-mediated B cell receptor stimulation, strain specific (autologous) neutralizing antibody (NAb) elicitation, and associated virus escape. In this virus/antibody ?arms race?, particular Env escape mutants are generated which, among the myriads of other Env variants present in infected individuals, are able to select for antibody lineages that acquire neutralization breadth. Although the principle of virus/antibody co-evolution has been established, the number, identity, succession, and cooperation of the particular Env variants that initiate and sustain bNAb lineage maturation have not been determined. It is also unclear to what extent the pathways of bNAb induction observed in one individual can be generalized to others. To address these questions, this HIVRAD team will test the scientific premise that simian-human immunodeficiency viruses (SHIVs) bearing HIV-1 Envs that engage V1V2 and V3-glycan bNAb unmutated common ancestor (UCA) and intermediate ancestor (IA) B cell receptors (BCR) in humans will bind related BCRs in rhesus macaques, leading to a recapitulation of virus/antibody coevolution and the development of NAb breadth and potency. We have already overcome a critical hurdle toward these objectives by generating primary and transmitted founder (TF) Env containing SHIVs, which replicate to high titers in rhesus macaques and yield patterns of early env evolution and autologous NAb escape that are astonishingly similar to those of humans infected with HIV-1 strains encoding the same Envs. These data suggest that B cell responses to a common Env antigen may, at least for some Envs, be deterministic and that by comparing the patterns of virus/antibody coevolution in outbred macaques and humans infected with the same Env expressing virus, it will be possible to identify common pathways of virus/BCR engagement and bNAb development that can be applied to vaccine design. The Virus and Antibody Gene Sequencing Core (Core B) will support this project by providing state-of-the-art sequencing services that will allow this HIVRAD consortium to dissect the mechanisms of SHIV elicited protective humoral immunity. Working closely with the Bioinformatics and Statistics Core (Core C) as well as Projects #1, #2 and #3, we will (i) characterize the evolving env quasispecies in SHIV infected macaques over time, and (ii) use Sanger and nextgen sequencing (NGS) methods to sequence antibody heavy (VHDJH) and light (VLJL) chain variable regions from clonal B cell cultures and rhesus memory B cells before and after SHIV infection and/or immunization. The goal is to understand the dynamic interactions between the evolving Env quasispecies and members of NAb and bNAb lineages to identify key Env escape mutations that reproducibly initiate and drive bNAb maturation.